The object of the proposed research is the identification and molecular analysis of virulence genes in Vibrio cholerae, the bacterium which causes the severe diarrheal disease cholera. Although the secretory diarrhea that is the hallmark of cholera can largely be explained by the actions of cholera toxin, many other gene products in this gram-negative organism contribute to its virulence. ToxR, a transmembrane DNA binding protein, is known to play a central role in the regulation of the expression of many virulence genes of V. cholerae including the genes encoding the biosynthesis of cholera toxin and the toxin co-regulated pilus. New virulence factors will be identified by finding novel ToxR-regulated genes. The process of identifying ToxR-regulated genes will involve two steps. First, a transposon which generates transcriptional fusions to bacterial genes will be used to construct libraries of V. cholerae strains with transposon insertion mutations. Second, these libraries will be screened to identify transposon insertions which are regulated by ToxR. Strains with transposon insertions in ToxR-regulated genes will be tested for altered virulence in mice. In strains which exhibit virulence defects in vivo, the linkage between the transposon insertion and the observed attenuated phenotype will be established. Once ToxR-regulated virulence genes have been identified they will be cloned and sequenced and homologies to known genes will be determined. Virulence genes will be identified in classical, El Tor and O139 strains of V. cholerae. In preliminary studies, the applicant has found that this st rat e gy can be successfully used to identify novel ToxR-regulated virulence genes in a classical strain of V. cholerae. This strategy not only yielded novel ToxR-activated virulence genes but also led tot he identification of ToxR-repressed virulence genes, a previously underscribed class of genes. Once new virulence genes are identified, cloned, and sequenced, deletions of these loci will facilitate the development of live attenuated cholera vaccine candidates. The promoters and/or parts of the amino acid sequences of the newly identified ToxR-regulated genes may be good candidates for use in the expression of heterologous antigens in cholera vaccines. Identifying V. cholerae virulence factors will provide a more complete molecular description of the pathogenesis of cholera and open new possibilities for vaccine strategies and possibly for drug design.